CD4+ Foxp3+ regulatory T (Treg) cells are important for maintaining immune tolerance, preventing autoimmune diseases, and fine-tuning of immune responses against invading pathogens. Although multiple transcription factors and signaling pathways that positively regulate natural Treg (nTreg) differentiation have been identified, littleis known about the negative regulatory mechanism involved in the process. Our recent studies showed that the SUMO E3 ligase PIAS1 (Protein Inhibitor of Activated PIAS1) restricts the differentiation of nTreg cells by maintaining a repressive chromatin state of the Foxp3 promoter. Pias1 deletion caused promoter demethylation, reduced H3K9 methylation, and enhanced promoter accessibility. Consistently, Pias1-/- mice displayed an increased nTreg population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of Treg cells. This application proposes experiments to directly address the deficiencies in our knowledge on Treg cells by studying the molecular mechanism and the biological significance of the newly identified PIAS1 epigenetic pathway in the differentiation and plasticity of Treg cells. Specifically, Aim 1 and Aim 2 will characterize the newly identified PIAS1 epigenetic pathway by testing novel aspects of the molecular mechanisms, including the exploration of previously unrecognized role of protein sumoylation in the epigenetic control of Treg cells and the analysis of specificity of the PIAS1 epigenetic pathway in Treg differentiation. Aim 3 will study the plasticity of Treg cells through an epigenetic approach. We will examine the reprogramming of Treg cells using novel animal models in which the epigenetic status of Treg cells is altered through Pias1 disruption. The successful completion of these studies will uncover novel molecular mechanisms involved in the negative regulation of Treg cells and will advance our ability to design effective therapeutic strategy for the treatment of autoimmune diseases.